Polypeptides having isoamylase activity and methods of use

ABSTRACT

The present invention relates to isolated polypeptides having isoamylase activity derived from  Dyella japonica  and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. The invention also relates to the use of said polypeptide having isoamylase activity for producing glucose syrup, fructose syrup, maltose syrup or maltitol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/859,993filed in Aug. 20, 2010, now U.S. Pat. No. 8,409,830, which claimspriority or the benefit under 35 U.S.C. 119 of European application no.09168390.4 filed Aug. 21, 2009 and U.S. provisional application No.61/237,805 filed Aug. 28, 2009, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference. Forcomplete information see the description.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides having isoamylaseactivity and isolated polynucleotides encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the polynucleotides as well as methods of producing andusing the polypeptides.

2. Description of the Related Art

Isoamylase is a debranching enzyme (E.C. 3.2.1.68) that hydrolyzes1,6-alpha-linkages in amylopectin, glycogen, and beta-limit dextrins.Amylopectin is partially degraded by alpha-amylase which hydrolyzes the1,4-alpha-glucosidic linkages into branched and linear oligosaccharidesthat results in the formation of alpha-limit dextrins. Unlikepullulanase, isoamylase has high activity towards amylopectin andglycogen and very low activity towards pullulan. Branchedoligosaccharides can be hydrolyzed into linear oligosaccharides bydebranching enzyme. The remaining linear oligosaccharides can be rapidlydepolymerized to D-glucose by glucoamylase.

U.S. Pat. No. 4,335,208 discloses a process of saccharifying starchhydrolysate by an enzyme mixture of glucoamylase and an acidophilicisoamylase derived from Pseudomonas amyloderamosa.

EP 1,002,062 concerns an isoamylase from Sulfolobus acidocaldarius andthe use thereof in a starch conversion process.

WO 2005/121305 (Novozymes) concerns a process for production of beerhaving a low content of carbohydrates which comprises; a) preparing amash in the presence of enzyme activities, b) filtering the mash toobtain a wort, and, c) fermenting said wort to obtain a beer, whereinthe enzyme activities comprise; an alpha-amylase, a glucoamylase and anisoamylase.

It is an object of the present invention to provide polypeptides havingisoamylase activity and polynucleotides encoding the polypeptides. Theinvention also provides uses of said isoamylase for syrup preparationand other related applications.

SUMMARY OF THE INVENTION

The present invention relates to an isolated polypeptide havingisoamylase activity, selected from the group consisting of:

(a1) a polypeptide comprising an amino acid sequence having at least93%, preferably at least 94%, more preferably at least 95%, morepreferably at least 96%, more preferably at least 97%, more preferablyat least 98%, even more preferably at least 99% identity to the maturepolypeptide of SEQ ID NO: 2;

(a2) a polypeptide comprising an amino acid sequence having at least94%, preferably at least 95%, more preferably at least 96%, morepreferably at least 97%, more preferably at least 98%, even morepreferably at least 99% identity to the mature polypeptide of SEQ ID NO:4;

(a3) a polypeptide comprising an amino acid sequence having at least93%, preferably at least 94%, more preferably at least 95%, morepreferably at least 96%, more preferably at least 97%, more preferablyat least 98%, even more preferably at least 99% identity to the maturepolypeptide of SEQ ID NO: 6;

(a4) a polypeptide comprising an amino acid sequence having at least90%, preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, even more preferably at least 99%identity to the mature polypeptide of SEQ ID NO: 8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7,(ii) the genomic DNA sequence comprising the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or(iii) a full-length complementary strand of (i) or (ii);

(c1) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 86%, preferably at least 88%, more preferablyat least 90%, more preferably at least 92%, even more preferably atleast 95%, most preferably at least 97%, and even most preferably atleast 99% identity to the mature polypeptide coding sequence of SEQ IDNO: 1;

(c2) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 86%, preferably at least 88%, more preferablyat least 90%, more preferably at least 92%, even more preferably atleast 95%, most preferably at least 97%, and even most preferably atleast 99% identity to the mature polypeptide coding sequence of SEQ IDNO: 3;

(c3) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 86%, preferably at least 88%, more preferablyat least 90%, more preferably at least 92%, even more preferably atleast 95%, most preferably at least 97%, and even most preferably atleast 99% identity to the mature polypeptide coding sequence of SEQ IDNO: 5;

(c4) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 84%, preferably at least 86%, more preferablyat least 87%, more preferably at least 88%, more preferably at least89%, more preferably at least 90%, more preferably at least 92%, evenmore preferably at least 95%, most preferably at least 97%, and evenmost preferably at least 99% identity to the mature polypeptide codingsequence of SEQ ID NO: 7; and

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNOs: 2, 4, 6 and/or 8.

The present invention also relates to an isolated polynucleotidecomprising a nucleotide sequence that encodes the polypeptide of theinvention. More specifically the invention in this aspect relates to anisolated polynucleotide encoding a polypeptide having isoamylaseactivity, obtained by:

(a) hybridizing a population of DNA under at least high stringencyconditions, preferably at least very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5 and/or SEQ ID NO: 7 (ii) the genomic DNA sequencecomprising the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7, or (iii) a full-lengthcomplementary strand of (i) or (ii);

(b) isolating the hybridized polynucleotide, which encodes a polypeptidehaving isoamylase activity.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, recombinant host cells comprising thepolynucleotides, and methods of producing a polypeptide havingisoamylase activity.

The present invention also relates to methods of producing beer andsyrups.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding such a polypeptide having isoamylase activity.

The present invention also relates to methods of producing such apolypeptide having isoamylase activity, comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingsuch a polypeptide having isoamylase activity under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.

DEFINITIONS

Isoamylase Activity:

The term “isoamylase activity” is defined herein as an enzyme havingglycogen alpha-1,6-glucanohydrolase activity (EC number 3.2.1.68)activity that catalyzes the hydrolysis of (1,6)-alpha-D-glucosidicbranch linkages in glycogen, amylopectin and their beta-limit dextrins.For purposes of the present invention, isoamylase activity is determinedaccording to the procedure as described in the “Materials &Methods”-section below under the heading “Determination of IsoamylaseActivity Units (IAU)”.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the isoamylase activity of the maturepolypeptide of SEQ ID NOS: 2, 4, 6, or 8, respectively.

Isolated Polypeptide:

The term “isolated polypeptide” as used herein refers to a polypeptidethat is isolated from a source. In a preferred aspect, the polypeptideis at least 1% pure, preferably at least 5% pure, more preferably atleast 10% pure, more preferably at least 20% pure, more preferably atleast 40% pure, more preferably at least 60% pure, even more preferablyat least 80% pure, and most preferably at least 90% pure, as determinedby SDS-PAGE. The term “pure” is in this context used in relation to thepolypeptide of the present invention and it is to be understood as anindication of how much other polypeptide material the polypeptide of thepresent invention is associated with.

Substantially Pure Polypeptide:

The term “substantially pure polypeptide” denotes herein a polypeptidepreparation that contains at most 10%, preferably at most 8%, morepreferably at most 6%, more preferably at most 5%, more preferably atmost 4%, more preferably at most 3%, even more preferably at most 2%,most preferably at most 1%, and even most preferably at most 0.5% byweight of other polypeptide material with which it is natively orrecombinantly associated. It is, therefore, preferred that thesubstantially pure polypeptide is at least 90% pure, preferably at least92% pure, more preferably at least 94% pure, more preferably at least95% pure, more preferably at least 96% pure, more preferably at least96% pure, more preferably at least 97% pure, more preferably at least98% pure, even more preferably at least 99%, most preferably at least99.5% pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptides of thepresent invention are preferably in a substantially pure form, i.e.,that the polypeptide preparation is essentially free of otherpolypeptide material with which it is natively or recombinantlyassociated. This can be accomplished, for example, by preparing thepolypeptide by well-known recombinant methods or by classicalpurification methods.

Mature Polypeptide:

The term “mature polypeptide” is defined herein as a polypeptide havingisoamylase activity that is in its final form following translation andany post-translational modifications, such as N-terminal processing,C-terminal truncation, glycosylation, phosphorylation, etc.

In a preferred aspect, the mature polypeptide is amino acids 1 to 750 ofSEQ ID NOS: 2, 4, 6 and 8, respectively, based on the SignaIP v 3.0program that predicts amino acids −26 to −1 of SEQ ID NOS: 2, 4, 6 and8, respectively, are signal peptides.

Mature Polypeptide Coding Sequence:

The term “mature polypeptide coding sequence” is defined herein as anucleotide sequence that encodes a mature polypeptide having isoamylaseactivity. In a preferred aspect, the mature polypeptide coding sequenceis nucleotides 79 to 2328 of SEQ ID NOS: 1, 3, 5 and 7, respectively,based on the SignaIP v 3.0 program that predicts nucleotides 1 to 78 ofSEQ ID NOS: 1, 3, 5, and 7, respectively, encode signal peptides.

Identity:

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Homologous Sequence:

The term “homologous sequence” is defined herein as a predicted proteinthat gives an E value (or expectancy score) of less than 0.001 in atfasty search (Pearson, W. R., 1999, in Bioinformatics Methods andProtocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) with theDyella japonica isoamylases (Accession Nos. DSM 22712, DSM 22713, DSM22714, and DSM 22715), respectively.

Polypeptide Fragment:

The term “polypeptide fragment” is defined herein as a polypeptidehaving one or more (several) amino acids deleted from the amino and/orcarboxyl terminus of the mature polypeptide of SEQ ID NOS: 2, 4, 6 or 8,respectively; or a homologous sequence thereof; wherein the fragment hasisoamylase activity. The term “fragment thereof” used in relation to apolypeptide is in the context of the present invention to be understoodas having the same meaning as “polypeptide fragment”.

Subsequence:

The term “subsequence” is defined herein as a nucleotide sequence havingone or more (several) nucleotides deleted from the 5′ and/or 3′ end ofthe mature polypeptide coding sequence of SEQ ID NOS: 1, 3, 5 or 7,respectively; or a homologous sequence thereof; wherein the subsequenceencodes a polypeptide fragment having isoamylase activity.

Allelic Variant:

The term “allelic variant” denotes herein any of two or more alternativeforms of a gene occupying the same chromosomal locus. Allelic variationarises naturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

Isolated Polynucleotide:

The term “isolated polynucleotide” as used herein refers to apolynucleotide that is isolated from a source. In a preferred aspect,the polynucleotide is at least 1% pure, preferably at least 5% pure,more preferably at least 10% pure, more preferably at least 20% pure,more preferably at least 40% pure, more preferably at least 60% pure,even more preferably at least 80% pure, and most preferably at least 90%pure, as determined by agarose electrophoresis. The term “pure” is inthis context used in relation to the polynucleotide of the presentinvention and it is to be understood as an indication of how much otherpolynucleotide material the polynucleotide of the present invention isassociated with.

Substantially Pure Polynucleotide:

The term “substantially pure polynucleotide” as used herein refers to apolynucleotide preparation free of other extraneous or unwantednucleotides and in a form suitable for use within genetically engineeredprotein production systems. Thus, a substantially pure polynucleotidecontains at most 10%, preferably at most 8%, more preferably at most 6%,more preferably at most 5%, more preferably at most 4%, more preferablyat most 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polynucleotidematerial with which it is natively or recombinantly associated. Asubstantially pure polynucleotide may, however, include naturallyoccurring 5′ and 3′ untranslated regions, such as promoters andterminators. It is preferred that the substantially pure polynucleotideis at least 90% pure, preferably at least 92% pure, more preferably atleast 94% pure, more preferably at least 95% pure, more preferably atleast 96% pure, more preferably at least 97% pure, even more preferablyat least 98% pure, most preferably at least 99%, and even mostpreferably at least 99.5% pure by weight. The polynucleotides of thepresent invention are preferably in a substantially pure form, i.e.,that the polynucleotide preparation is essentially free of otherpolynucleotide material with which it is natively or recombinantlyassociated. The polynucleotides may be of genomic, cDNA, RNA,semisynthetic, or synthetic origin, or any combinations thereof.

Coding Sequence:

When used herein the term “coding sequence” means a nucleotide sequence,which directly specifies the amino acid sequence of its protein product.The boundaries of the coding sequence are generally determined by anopen reading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant nucleotide sequence.

Nucleic Acid Construct:

The term “nucleic acid construct” as used herein refers to a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which is modified to contain segments ofnucleic acids in a manner that would not otherwise exist in nature orwhich is synthetic. The term nucleic acid construct is synonymous withthe term “expression cassette” when the nucleic acid construct containsthe control sequences required for expression of a coding sequence ofthe present invention.

Control Sequences:

The term “control sequences” is defined herein to include all componentsnecessary for the expression of a polynucleotide encoding a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleotide sequence encoding the polypeptide or native or foreignto each other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably Linked:

The term “operably linked” denotes herein a configuration in which acontrol sequence is placed at an appropriate position relative to thecoding sequence of the polynucleotide sequence such that the controlsequence directs the expression of the coding sequence of a polypeptide.

Expression:

The term “expression” includes any step involved in the production ofthe polypeptides including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression Vector:

The term “expression vector” is defined herein as a linear or circularDNA molecule that comprises a polynucleotide encoding a polypeptide ofthe present invention and is operably linked to additional nucleotidesthat provide for its expression.

Host Cell:

The term “host cell”, as used herein, includes any cell type that issusceptible to transformation, transfection, transduction, and the likewith a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification:

The term “modification” means herein any chemical modification of thepolypeptide consisting of the mature polypeptide of SEQ ID NOS: 2, 4, 6or 8, respectively; or a homologous sequence thereof; as well as geneticmanipulation of the DNA encoding such a polypeptide. The modificationcan be a substitution, a deletion and/or an insertion of one or more(several) amino acids as well as replacements of one or more (several)amino acid side chains.

Artificial Variant:

When used herein, the term “artificial variant” means a polypeptidehaving isoamylase activity produced by an organism expressing a modifiedpolynucleotide sequence of the mature polypeptide coding sequence of SEQID NOS: 1, 3, 5 or 7; or a homologous sequence thereof. The modifiednucleotide sequence is obtained through human intervention bymodification of the polynucleotide sequence disclosed in SEQ ID NOS: 1,3, 5 and/or 7; or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Isoamylase Activity

In a first aspect, the present invention relates to an isolatedpolypeptide having isoamylase activity, selected from the groupconsisting of:

(a1) a polypeptide comprising an amino acid sequence having at least93%, more preferably at least 94%, more preferably at least 95%, morepreferably at least 96%, more preferably at least 97%, more preferablyat least 98%, even more preferably at least 99% identity to the maturepolypeptide of SEQ ID NO: 2;

(a2) a polypeptide comprising an amino acid sequence having at least94%, more preferably at least 95%, more preferably at least 96%, morepreferably at least 97%, more preferably at least 98%, even morepreferably at least 99% identity to the mature polypeptide of SEQ ID NO:4;

(a3) a polypeptide comprising an amino acid sequence having at least93%, more preferably at least 94%, more preferably at least 95%, morepreferably at least 96%, more preferably at least 97%, more preferablyat least 98%, even more preferably at least 99% identity to the maturepolypeptide of SEQ ID NO: 6;

(a4) a polypeptide comprising an amino acid sequence having at least90%, preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, even more preferably at least 99%identity to the mature polypeptide of SEQ ID NO: 8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7(ii) the genomic DNA sequence comprising the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or(iii) a full-length complementary strand of (i) or (ii);

(c1) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 86%, preferably at least 88%, more preferablyat least 90%, more preferably at least 92%, even more preferably atleast 95%, most preferably at least 97%, and even most preferably atleast 99% identity to the mature polypeptide coding sequence of SEQ IDNO: 1

(c2) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 86%, preferably at least 88%, more preferablyat least 90%, more preferably at least 92%, even more preferably atleast 95%, most preferably at least 97%, and even most preferably atleast 99% identity (hereinafter “homologous polypeptides”) to the maturepolypeptide coding sequence of SEQ ID NO: 3;

(c3) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 86%, preferably at least 88%, more preferablyat least 90%, more preferably at least 92%, even more preferably atleast 95%, most preferably at least 97%, and even most preferably atleast 99% identity to the mature polypeptide coding sequence of SEQ IDNO: 5;

(c4) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 84%, preferably at least 86%, more preferablyat least 87%, more preferably at least 88%, more preferably at least89%, more preferably at least 90%, more preferably at least 92%, evenmore preferably at least 95%, most preferably at least 97%, and evenmost preferably at least 99% identity to the mature polypeptide codingsequence of SEQ ID NO: 7; and

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNOS: 2, 4, 6 and/or 8.

In a preferred aspect, the homologous polypeptides have an amino acidsequence that differs by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NOS:2, 4, 6 and/or 8.

In one embodiment the polypeptide of the present invention comprises anamino acid sequence, wherein said amino acid sequence consists of theamino acid sequence of SEQ ID NOS: 2, 4, 6, 8 or an amino acid sequencethat differs by 1-70 amino acids, such as 1-65 amino acids, or 1-60amino acids, or 1-55 amino acids, or 1-50 amino acids, or 1-45 aminoacids, or 1-40 amino acids, or 1-35 amino acids, or 1-30 amino acids, or1-25 amino acids, or 1-20 amino acids, or 1-15 amino acids, or 1-10amino acids, or 1-9 amino acids, or 1-8 amino acids, or 1-7 amino acids,or 1-6 amino acids, or 1-5 amino acids, or 1-4 amino acids, or 1-3 aminoacids, or 1-2 amino acids or 1 amino acid from the amino acid sequenceof SEQ ID NOS: 2, 4, 6 and/or 8, wherein the term “differ” means thatthe given number of amino acids have been substituted, deleted and/orinserted when compared to the amino acid sequence of SEQ ID NOS: 2, 4,6, 8.

In another embodiment the polypeptide of the present invention comprisesan amino acid sequence, wherein said amino acid sequence consists of themature polypeptide of SEQ ID NOS: 2, 4, 6, and 8 and/or an amino acidsequence that differs by 1-70 amino acids, such as 1-65 amino acids, or1-60 amino acids, or 1-55 amino acids, or 1-50 amino acids, or 1-45amino acids, or 1-40 amino acids, or 1-35 amino acids, or 1-30 aminoacids, or 1-25 amino acids, or 1-20 amino acids, or 1-15 amino acids, or1-10 amino acids, or 1-9 amino acids, or 1-8 amino acids, or 1-7 aminoacids, or 1-6 amino acids, or 1-5 amino acids, or 1-4 amino acids, or1-3 amino acids, or 1-2 amino acids or 1 amino acid from the maturepolypeptide of SEQ ID NOS: 2, 4, 6 and/or 8, wherein the term “differ”means that the given number of amino acids have been substituted,deleted and/or inserted when compared to the amino acid sequence of SEQID NOS: 2, 4, 6, 8.

In another embodiment the polypeptide of the present invention comprisesan amino acid sequence, wherein said amino acid sequence consists ofamino acids 1 to 750 of SEQ ID NOS: 2, 4, 6, 8 and/or an amino acidsequence that differs by 1-70 amino acids, such as 1-65 amino acids, or1-60 amino acids, or 1-55 amino acids, or 1-50 amino acids, or 1-45amino acids, or 1-40 amino acids, or 1-35 amino acids, or 1-30 aminoacids, or 1-25 amino acids, or 1-20 amino acids, or 1-15 amino acids, or1-10 amino acids, or 1-9 amino acids, or 1-8 amino acids, or 1-7 aminoacids, or 1-6 amino acids, or 1-5 amino acids, or 1-4 amino acids, or1-3 amino acids, or 1-2 amino acids or 1 amino acid from amino acids 1to 750 of SEQ ID NOS: 2, 4, 6 and/or 8, wherein the term “differ” meansthat the given number of amino acids have been substituted, deletedand/or inserted when compared to the amino acid sequence of SEQ ID NOS:2, 4, 6, 8.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NOS: 2, 4, 6 and/or 8 or variants thereof; orfragments thereof having isoamylase activity. The variants may inparticular be an allelic variant, an artificial variant or a variantcomprising a substitution, deletion, and/or insertion of one or more(several) amino acids of the mature polypeptide of SEQ ID NOS: 2, 4, 6and/or 8. In a preferred aspect, the polypeptide comprises the aminoacid sequence of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferredaspect, the polypeptide comprises the mature polypeptide of SEQ ID NO:2, 4, 6, and/or 8. In another preferred aspect, the polypeptidecomprises amino acids 1 to 750 of SEQ ID NOS: 2, 4, 6 and/or 8respectively, or a variant thereof; or fragments thereof havingisoamylase activity. The variants may in particular be an allelicvariant, an artificial variant or a variant comprising a substitution,deletion, and/or insertion of one or more (several) amino acids of themature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferredaspect, the polypeptide comprises amino acids 1 to 750 of SEQ ID NOS: 2,4, 6 and/or 8. In another preferred aspect, the polypeptide consists ofthe amino acid sequence of SEQ ID NOS: 2, 4, 6 and/or 8; or variantsthereof; or fragments thereof having isoamylase activity. The variantsmay in particular be an allelic variant, an artificial variant or avariant comprising a substitution, deletion, and/or insertion of one ormore (several) amino acids of the mature polypeptide of SEQ ID NOS: 2,4, 6 and/or 8. In another preferred aspect, the polypeptide consists ofthe amino acid sequence of SEQ ID NOS: 2, 4, 6 and/or 8. In anotherpreferred aspect, the polypeptide consists of the mature polypeptide ofSEQ ID NOS: 2, 4, 6 and/or 8. In another preferred aspect, thepolypeptide consists of amino acids 1 to 750 of SEQ ID NOS: 2, 4, 6and/or 8; or a variant thereof; or a fragment thereof having isoamylaseactivity. The variant may in particular be an allelic variant, anartificial variant or a variant comprising a substitution, deletion,and/or insertion of one or more (several) amino acids of the maturepolypeptide of SEQ ID NOS: 2, 4, 6 and/or 8. In another preferredaspect, the polypeptide consists of amino acids 1 to 750 of SEQ ID NOS:2, 4, 6 and/or 8.

In a second aspect, the present invention relates to an isolatedpolynucleotide encoding polypeptides having isoamylase activity,obtained by:

(a) hybridizing a population of DNA under at least high stringencyconditions, preferably at least very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5 and/or SEQ ID NO: 7 (ii) the genomic DNA sequencecomprising the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7, or (iii) a full-lengthcomplementary strand of (i) or (ii);

(b) isolating the hybridized polynucleotide, which encodes a polypeptidehaving isoamylase activity.

Therefore, the invention relates to isolated polypeptides havingisoamylase activity that are encoded by polynucleotides that hybridizeunder preferably high stringency conditions, preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, 3, 5, and/or 7, (ii) the genomic DNA sequence comprisingthe mature polypeptide coding sequence of SEQ ID NO: 1, 3, 5 and/or 7,(iii) a subsequence of (i) or (ii), or (iv) a full-length complementarystrand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, New York). A subsequence of the mature polypeptide codingsequence of SEQ ID NOS: 1, 3, 5 and/or 7, respectively, contain at least100 contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmenthaving isoamylase activity. In a preferred aspect, the complementarystrand is the full-length complementary strand of the mature polypeptidecoding sequence of SEQ ID NOS: 1, 3, 5 and/or 7.

The nucleotide sequence of SEQ ID NOS: 1, 3, 5 and/or 7; or asubsequence thereof; as well as the amino acid sequence of SEQ ID NOS:2, 4, 6 and/or 8; or fragments thereof; may be used to design nucleicacid probes to identify and clone DNA encoding polypeptides havingisoamylase activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 14, preferably at least 25, more preferably at least 35, andmost preferably at least 70 nucleotides in length. It is, however,preferred that the nucleic acid probe is at least 100 nucleotides inlength.

For example, the nucleic acid probe may be at least 200 nucleotides,preferably at least 300 nucleotides, more preferably at least 400nucleotides, or most preferably at least 500 nucleotides in length. Evenlonger probes may be used, e.g., nucleic acid probes that are preferablyat least 600 nucleotides, more preferably at least 700 nucleotides, evenmore preferably at least 800 nucleotides, or most preferably at least900 nucleotides in length. Both DNA and RNA probes can be used. Theprobes are typically labeled for detecting the corresponding gene (forexample, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA library prepared from such other strains may, therefore,be screened for DNA that hybridizes with the probes described above andencodes a polypeptide having isoamylase activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that is homologous with SEQ ID NOS: 1, 3, 5 and/or 7; orsubsequences thereof; the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NOS:1, 3, 5 and/or 7; the genomic DNA sequence comprising the maturepolypeptide coding sequence of SEQ ID NOS: 1, 3, 5, and/or 7; itsfull-length complementary strands; or a subsequence thereof; under highto very high stringency conditions. Molecules to which the nucleic acidprobe hybridizes under these conditions can be detected using, forexample, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NOS: 1, 3, 5 and/or 7. In another preferredaspect, the nucleic acid probe is nucleotides 79 to 2328 of SEQ ID NOS:1, 3, 5 and/or 7, respectively. In another preferred aspect, the nucleicacid probe is a polynucleotide sequence that encodes the polypeptide ofSEQ ID NOS: 2, 4, 6 and/or 8, or a subsequence thereof. In anotherpreferred aspect, the nucleic acid probe is SEQ ID NOS: 1, 3, 5 and/or7. In another preferred aspect, the nucleic acid probe is one of thepolynucleotide sequences contained in and obtainable from depositedstrains Dyella japonica DSM 22712, Dyella japonica DSM 22713, Dyellajaponica DSM 22714 and/or Dyella japonica DSM 22715, wherein thepolynucleotide sequence thereof encodes polypeptides having isoamylaseactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding region contained in and obtainable fromdeposited strains Dyella japonica DSM 22712, Dyella japonica DSM 22713,Dyella japonica DSM 22714 and/or Dyella japonica DSM 22715.

For long probes of at least 100 nucleotides in length, high and veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micro g/ml sheared anddenatured salmon sperm DNA, and 50% formamide for high and very highstringencies, following standard Southern blotting procedures for 12 to24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at 65° C. (high stringency), and most preferably at70° C. (very high stringency).

For short probes that are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(n), using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes that are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides having isoamylase activity encoded by polynucleotidescomprising or consisting of

(c1) a nucleotide sequence having at least 86%, preferably at least 88%,more preferably at least 90%, more preferably at least 92%, even morepreferably at least 95%, most preferably at least 97%, and even mostpreferably at least 99% identity to the mature polypeptide codingsequence of SEQ ID NO: 1;

(c2) a nucleotide sequence having at least 86%, preferably at least 88%,more preferably at least 90%, more preferably at least 92%, even morepreferably at least 95%, most preferably at least 97%, and even mostpreferably at least 99% identity to the mature polypeptide codingsequence of SEQ ID NO: 3;

(c3) a nucleotide sequence having at least 86%, preferably at least 88%,more preferably at least 90%, more preferably at least 92%, even morepreferably at least 95%, most preferably at least 97%, and even mostpreferably at least 99% identity to the mature polypeptide codingsequence of SEQ ID NO: 5;

(c4) a nucleotide sequence having at least 84%, preferably at least 86%,more preferably at least 87%, more preferably at least 88%, morepreferably at least 89%, more preferably at least 90%, more preferablyat least 92%, even more preferably at least 95%, most preferably atleast 97%, and even most preferably at least 99% identity to the maturepolypeptide coding sequence of SEQ ID NO: 7.

In a fourth aspect, the present invention relates to artificial variantscomprising a substitution, deletion, and/or insertion of one or more (orseveral) amino acids of the mature polypeptide of SEQ ID NOS: 2, 4, 6and/or 8; or homologous sequences thereof. Preferably, amino acidchanges are of a minor nature, that is conservative amino acidsubstitutions or insertions that do not significantly affect the foldingand/or activity of the protein; small deletions, typically of one toabout 30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up toabout 20-25 residues; or a small extension that facilitates purificationby changing net charge or another function, such as a poly-histidinetract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

The amino acid substitutions in the wild-type polypeptide include bothsubstitutions with the 20 standard amino acids but also non-standardamino acids (such as 4-hydroxyproline, 6-N-methyl lysine,2-aminoisobutyric acid, isovaline, and alpha-methyl serine). A limitednumber of non-conservative amino acids, amino acids that are not encodedby the genetic code, and unnatural amino acids may be substituted foramino acid residues. “Unnatural amino acids” have been modified afterprotein synthesis, and/or have a chemical structure in their sidechain(s) different from that of the standard amino acids. Unnaturalamino acids can be chemically synthesized, and preferably, arecommercially available, and include pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-methylproline, and3,3-dimethylproline.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,isoamylase activity) to identify amino acid residues that are criticalto the activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides that are related to apolypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8, is10, preferably 9, more preferably 8, more preferably 7, more preferablyat most 6, more preferably 5, more preferably 4, even more preferably 3,most preferably 2, and even most preferably 1.

Sources of Polypeptides Having Isoamylase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.Examples of sources from which a polypeptide of the present inventionmay be obtained include but are not limited to any of the followingdescribed below.

A polypeptide having isoamylase activity of the present invention may bea bacterial polypeptide. For example, the polypeptide may be a grampositive bacterial polypeptide such as a Bacillus, Streptococcus,Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,Clostridium, Geobacillus, or Oceanobacillus polypeptide havingisoamylase activity, or a Gram negative bacterial polypeptide such as aDyella, Fulvimonas, Frateuria and Rhodanobacter E. coli, Pseudomonas,Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium,Ilyobacter, Neisseria, or Ureaplasma polypeptide having isoamylaseactivity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having isoamylase activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having isoamylaseactivity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingisoamylase activity.

In another preferred aspect, the polypeptide is a Pseudomonasamyloderamosa polypeptide, e.g., the one descried in (Harada et al.,1968; Sugimoto et al., 1974) having isoamylase activity.

In another preferred aspect, the polypeptide is a Frateuria aurantiapolypeptide having isoamylase activity.

In another preferred aspect, the polypeptide is a Dyella japonica andDyella koreensis polypeptide having isoamylase activity.

In a more preferred aspect, the polypeptide is a Dyella japonicapolypeptide having isoamylase activity. In a most preferred aspect, thepolypeptide is a Dyella japonica DSM 22712 DSM 22713, DSM 22714 and/orDSM 22715 polypeptide having isoamylase activity, e.g., the polypeptidescomprising the mature polypeptides of SEQ ID NOS: 2, 4, 6 or 8,respectively.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques thatare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having isoamylase activity from the fusion protein. Examplesof cleavage sites include, but are not limited to, a Kex2 site thatencodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind. Microbiol.Biotechnol. 3: 568-76; Svetina et al., 2000, J. Biotechnol. 76: 245-251;Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493;Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al.,1991, Biotechnology 9: 378-381), an Ile-(Glu or Asp)-Gly-Arg site (SEQID NO: 21), which is cleaved by a Factor Xa protease after the arginineresidue (Eaton et al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site (SEQ ID NO: 22), which is cleaved by anenterokinase after the lysine (Collins-Racie et al., 1995, Biotechnology13: 982-987); a His-Tyr-Glu site or His-Tyr-Asp site, which is cleavedby Genenase I (Carter et al., 1989, Proteins: Structure, Function, andGenetics 6: 240-248); a Leu-Val-Pro-Arg-Gly-Ser site (SEQ ID NO: 23),which is cleaved by thrombin after the Arg (Stevens, 2003, DrugDiscovery World 4: 35-48); a Glu-Asn-Leu-Tyr-Phe-Gln-Gly site (SEQ IDNO: 24), which is cleaved by TEV protease after the Gln (Stevens, 2003,supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site (SEQ ID NO: 25),which is cleaved by a genetically engineered form of human rhinovirus 3Cprotease after the Gln (Stevens, 2003, supra).

Polynucleotides

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that encodepolypeptides having isoamylase activity of the present invention.

The polypeptide of the present invention may in particular be encoded bya polynucleotide comprising or consisting of any of the nucleotidesequences of SEQ ID NOS: 1, 3, 5 and/or 7; or a subsequence thereofencoding a fragment having isoamylase activity.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NOS: 1, 3, 5 and/or 7, respectively. In another more preferredaspect, the nucleotide sequence comprises or consists of the sequencecontained in the deposited strain: Dyella japonica NN060811 (DSM 22712);Dyella japonica NN060812 (DSM 22713); Dyella japonica NN060813 (DSM22714); Dyella japonica NN060814 (DSM 22715).

In another preferred aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NOS: 1, 3,5 and/or 7.

The present invention also encompasses nucleotide sequences that encodepolypeptides comprising or consisting of the amino acid sequence of SEQID NOS: 2, 4, 6 and/or 8, respectively, or the mature polypeptidethereof, which differ from SEQ ID NOS: 1, 3, 5 and/or 7 or the maturepolypeptide coding sequences thereof by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NOS: 1, 3, 5 and/or 7 that encode fragments of SEQ ID NOS: 2, 4, 6and/or 8, respectively, that have isoamylase activity.

The present invention also relates to mutant polynucleotides comprisingor consisting of at least one mutation in the mature polypeptide codingsequence of SEQ ID NOS: 1, 3, 5 and/or 7, in which the mutant nucleotidesequence encodes the mature polypeptide of SEQ ID NOS: 2, 4, 6 and/or 8.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Dyella, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NOS: 1, 3,5 and/or 7, respectively.

The present invention also relates to isolated polynucleotides encodingpolypeptides having isoamylase activity, obtained by:

(a) hybridizing a population of DNA under at least high stringencyconditions, preferably at least very high stringency conditions, with(i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5 and/or SEQ ID NO: 7 (ii) the genomic DNA sequencecomprising the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7, or (iii) a full-lengthcomplementary strand of (i) or (ii);

(b) isolating the hybridized polynucleotide, which encodes a polypeptidehaving isoamylase activity.

The isolated polynucleotide of the invention may be the maturepolypeptide coding sequence in nucleotides 79-2328 of SEQ ID NOS: 1, 3,5 and/or 7.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the mature polypeptide coding sequenceof SEQ ID NOS: 1, 3, 5 and/or 7, e.g., a subsequence thereof, and/or byintroduction of nucleotide substitutions that do not give rise toanother amino acid sequence of the polypeptide encoded by the nucleotidesequence, but which correspond to the codon usage of the host organismintended for production of the enzyme, or by introduction of nucleotidesubstitutions that may give rise to a different amino acid sequence. Fora general description of nucleotide substitution, see, e.g., Ford etal., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, supra). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for isoamylase activity toidentify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labeling (see, e.g., de Vos et al., 1992, supra; Smith etal., 1992, supra; Wlodaver et al., 1992, supra).

In a preferred aspect, the complementary strand is the full-lengthcomplementary strand of the mature polypeptide coding sequence of SEQ IDNOS: 1, 3, 5 and/or 7.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more (several) control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The present invention also relates to a nucleic acid constructcomprising an isolated polynucleotide comprising a nucleotide sequencethat encodes the polypeptide of the present invention, operably linkedto one or more (several) control sequences that direct the production ofthe polypeptide in an expression host.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Combinations of promotersmay also be used, such as the triple promoter system described in WO99/43835 or example 1 of the present application, which consists of thepromoters from Bacillus licheniformis alpha-amylase gene (amyL),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillusthuringiensis cryIIIA promoter including stabilizing sequence. Furtherpromoters are described in “Useful proteins from recombinant bacteria”in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989,supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Examples of terminators for bacterial host cells include but are notlimited to those obtained from the genes of Bacillus licheniformis BPN'and Bacillus licheniformis amyl.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding sequencenaturally linked in translation reading frame with the segment of thecoding sequence that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingsequence that is foreign to the coding sequence. The foreign signalpeptide coding sequence may be required where the coding sequence doesnot naturally contain a signal peptide coding sequence. Alternatively,the foreign signal peptide coding sequence may simply replace thenatural signal peptide coding sequence in order to enhance secretion ofthe polypeptide. However, any signal peptide coding sequence thatdirects the expressed polypeptide into the secretory pathway of a hostcell of choice, i.e., secreted into a culture medium, may be used in thepresent invention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), Bacillus clausii alcaline protease (aprH) and Bacillussubtilis prsA. Further signal peptides are described by Simonen andPalva, 1993, Microbiological Reviews 57: 109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

In a preferred aspect, the signal peptide comprises or consists of aminoacids −26 to −1 of SEQ ID NOS: 2, 4, 6 and/or 8, respectively. Inanother preferred aspect, the signal peptide coding sequence comprisesor consists of nucleotides 1 to 78 of SEQ ID NOS: 1, 3, 6 and/or 8,respectively.

The control sequence may also be a propeptide coding sequence that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding sequence may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, xyl and trp operator systems.

In yeast, the ADH2 system or GAL1 system may be used. In filamentousfungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter may be used asregulatory sequences. Other examples of regulatory sequences are thosethat allow for gene amplification. In eukaryotic systems, theseregulatory sequences include the dihydrofolate reductase gene that isamplified in the presence of methotrexate, and the metallothionein genesthat are amplified with heavy metals. In these cases, the nucleotidesequence encoding the polypeptide would be operably linked with theregulatory sequence.

Expression Vectors

The present invention also related to a recombinant expression vectorcomprising a nucleic acid construct of the present invention.

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector that may include one or more(several) convenient restriction sites to allow for insertion orsubstitution of the nucleotide sequence encoding the polypeptide at suchsites. Alternatively, a polynucleotide sequence of the present inventionmay be expressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors of the present invention preferably contain one or more(several) selectable markers that permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance.

Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3. Selectable markers for use in a filamentous fungal hostcell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell are theamdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae andthe bar gene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity to the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of the gene product. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisingan isolated polynucleotide of the present invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a polynucleotide of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

The present invention also relates to a recombinant host cell comprisinga nucleic acid construct of the present invention.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive bacterium or agram-negative bacterium. Gram-positive bacteria include, but are notlimited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram-negative bacteria include, but are not limited to,E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Dyella. In a morepreferred aspect, the cell is Dyella japonica. In a most preferredaspect, the cell is Dyella japonica DSM 22712, Dyella japonica DSM22713, Dyella japonica DSM 22714 and/or Dyella japonica DSM 22715,respectively.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell, as described herein, under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.

The present invention also relates to a method of producing thepolypeptide of the present invention, comprising: (a) cultivating a hostcell comprising a nucleic acid construct comprising a nucleotidesequence encoding the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. Thepresent invention also relates to methods of producing a polypeptide ofthe present invention, comprising: (a) cultivating a recombinant hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNOS: 1, 3, 5 and/or 7, wherein the mutant nucleotide sequence encodes apolypeptide that comprises or consists of the mature polypeptide of SEQID NOS: 2, 4, 6 and/or 8, respectively, and (b) recovering thepolypeptide.

The present invention also relates to a method of producing apolypeptide of the present invention, comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. In the productionmethods of the present invention, the cells are cultivated in a nutrientmedium suitable for production of the polypeptide using methods wellknown in the art. For example, the cell may be cultivated by shake flaskcultivation, and small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

The polypeptides of the present invention may in a particular embodimentbe purified by starch affinity chromatography, e.g., by usingamylase-agarose as the affinity material. Methods of performing thisinclude but are not limited to the method described below in example 2.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotideencoding a polypeptide having isoamylase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The present invention also relates to a transgenic plant, plant part orplant cell transformed with a polynucleotide encoding a polypeptide ofthe present invention.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more (several) expression constructs encoding apolypeptide of the present invention into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptidehaving isoamylase activity of the present invention under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theisoamylase activity of the composition has been increased, e.g., with anenrichment factor of at least 1.1.

The composition may comprise an isoamylase of the present invention asthe major enzymatic component, e.g., a mono-component composition. In anembodiment the composition may further comprise one or moreglucoamylases, in particular derived from a strain of the genusAspergillus, Trichoderma, Talaromyces or Trametes, including Aspergillusniger, Talaromyces emersonii, Trametes cingulata, and Trichodermareesei. In another embodiment the composition further comprising one ormore additional enzymes selected from the group of proteases,alpha-amylases, beta-amylases, maltogenic amylases, alpha-glucosidases,pullulanases, hexosyltransferase and branching enzymes.

The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

The dosage of the polypeptide composition of the invention and otherconditions under which the composition is used may be determined on thebasis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having isoamylase activity, or compositions thereof.

In one embodiment an isoamylase of the invention may be used inbeverage, such as beer, mashing process.

In an embodiment an isoamylase of the invention may be used in theproduction of food ingredients from starch. Isoamylase are typicallyused in combination with other starch-hydrolysing enzymes, such asalpha-amylase, beta-amylase and glucoamylase. Depending on the specificapplication, isoamylase is used at levels between 50-5000 IAU per gramstarch.

In the production of glucose syrup from starch, starch is liquefiedusing alpha-amylase, such as bacterial alpha-amylase, e.g., one or moreBacillus alpha-amylase. Subsequently, glucoamylase may be added toconvert the starch hydrolysate to glucose syrup. The addition ofisoamylase of the invention may then result in syrup with higher glucosecontent while reducing the amount of added glucoamylase.

Thus the present invention also relates to the use of an isoamylase ofthe present invention or a composition of the present invention forproducing a glucose syrup.

The present invention also relates to the use of an isoamylase of thepresent invention or a composition of the present invention for aprocess for producing high fructose syrup process, in particular forproducing HFCS.

Specific examples of the methods contemplated for high DX glucose syrup(e.g., 97-98 DX) production using mixtures of glucoamylase andisoamylase are described in U.S. Pat. No. 4,335,208, in particularexamples 1, 3, 4, 5, 6 and 7, which is hereby incorporated by reference.

The present invention also relates to the use of an isoamylase of thepresent invention or a composition of the present invention in a processfor producing maltose or maltitol.

An isoamylase of the invention may also be used in the production ofmaltose and maltitol.

In such embodiment the isoamylase is added to liquefied starch afterbeing subjected to alpha-amylase. At the same time, beta-amylase may beadded. The hydrolysis may be carried out at temperature around 50-60°C., e.g., at 50-55° C., and a pH of around pH 4-6, e.g., about 5.0). Theresulting high maltose syrup may subsequently be purified andconcentrated and subjected to crystallization to obtain crystallinemaltose. Maltose may then be converted to maltitol by the catalytichydrogenation. Maltitol is used as a sugar substitute in the productionof non-cariogenic hard candies, chewing gum, and other confectionary.

An isoamylase of the invention may also be used together withcyclodextrin glucanotransferase (CGTase), malto-oligosyl trehalosesynthase, and malto-oligosyl trehalose trehalohydrolase in theproduction of a disaccharide trehalose from liquefied starch. Thereaction product is trehalose syrup, which is subsequently purified andconcentrated. Trehalose is used in food (for example, in bakery goods,beverages, confectionery, and breakfast cereals) as a texturizer,stabilizer, humectant, and sweetener.

An isoamylase of the invention may also be used in conjunction withCGTase to enhance the production of cyclodextrins from starch. Forexample, a 90% yield of beta-cyclodextrin from amylopectin was obtainedby applying a mixture of isoamylase, CGTase, and cyclodecanone at aroundpH 6 and 25° C. Cyclodecanone is a complexant that forms an insolubleinclusion complex with a cyclodextrin molecule. Cyclodextrins are usedas encapsulating agents for food additives, flavours, and vitamins.

Generally isoamylase activity is preferably used in amounts of 1 to1,000,000,000 IAU/kg DS, more preferably 10 to 100,000,000 IAU/kg DS,even more preferably 100 to 10,000,000 IAU/kg DS, and most preferably50,000 to 5,000,000 IAU/kg DS or 0.001 mg to 100,000 mg EP/kg DS,preferably in the amount of 0.01 mg to 10,000 mg EP/kg DS, morepreferably in the amount of 0.1 mg to 1,000 mg EP/kg DS, most preferablyin the amount of 1 mg to 100 mg EP/kg DS.

The invention also relates to a method of producing glycogen using anisoamylase, branching enzyme and amylomaltase (EC 2.4.1.25), e.g., asdescribed by Kajuura et al., 2008, Biocatalysis and Biotransformation26(1-2): 133-140.

Materials & Methods

Materials

Alpha-Amylase LS:

Blend of 2 parts hybrid alpha-amylase comprising 445 C-terminal aminoacid residues of the Bacillus licheniformis alpha-amylase (shown in SEQID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues ofthe alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQID NO: 5 of WO 99/19467), with the following substitution:G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S and 1 part Bacillusstearothermophilus alpha-amylase with the following mutations:I181*+G182*+N193F.Glucoamylase AN:Glucoamylase from Aspergillus niger.Glucoamylase T:Glucoamylase derived from Talaromyces emersonii disclosed in SEQ ID NO:7 in WO 99/28448 and available from Novozymes NS, Denmark.Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), lnffenstr. 7 B, D-38124 Braunschweig, Germany,and given the following accession number:

Deposit Accession Number Date of Deposit Dyella japonica NN060811 DSM22712 Jun. 24, 2009 Dyella japonica NN060812 DSM 22713 Jun. 24, 2009Dyella japonica NN060813 DSM 22714 Jun. 24, 2009 Dyella japonicaNN060814 DSM 22715 Jun. 24, 2009

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Deposit Country of Origin Year Dyella japonica NN060811 Denmark 2007Dyella japonica NN060812 China 2007 Dyella japonica NN060813 China 2007Dyella japonica NN060814 China 2007Methods:Determination of Isoamylase Activity Units (IAU)

The debranching of starch is measured as an increase in iodine bindingand thereby increases in blue color due to the fact that linear glucansbinds iodine more strongly than branched glucans (amylopectin).

1 unit (U) of isoamylase activity (IAU) is the amount of enzyme whichcauses an increase in absorbency of 0.01 per minute at 610 nm at 40° C.,pH 4.5.

Substrate: 1% waxy corn starch (amylopectin) in 75 mM Na-acetate pH 4.5,2 mM CaCl₂. The substrate should be boiled for 10-15 minutes tosolubilise the starch.

Iodine/stop reagent: 10 mM I₂/KI in MQ H₂O

Sulphuric acid solution: 20 mM H₂SO₄ in MQ H₂O

Assay Procedure:

Mix

1. 600 microL substrate

2. 100 microL enzyme solution (include a control which should be 100microL MQ H₂O)

3. Incubate at 40° C., 30 minutes

4. Withdraw 50 microL after 30 minutes into an eppendorf tube whichalready contains 50 microL of the iodine solution.

5. Subsequently add 1.5 mL of the sulphuric acid solution and transfer200 microL to a MTP plate.

6. After 5 minutes read the absorbance at 610 nm

Activity Calculation:U/mL=OD610 (sample)−OD610 (control)/0.01/30 minutes/50 microL*1000microLGlucoamylase Activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL Color reaction: GlucDH: 430 U/LMutarotase: 9 U/L NAD: 0.21 mM Buffer: phosphate 0.12M; 0.15M NaCl pH:7.60 ± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesWavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes NS, Denmark, which folderis hereby included by reference.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Example 1

Cloning and Expression of Four Isoamylases from Dyella japonica inBacillus subtilis

A linear integration vector-system was used for the expression cloningof four isoamylase genes. The linear integration construct was a PCRfusion product made by fusion of each gene between two Bacillus subtilishomologous chromosomal regions along with a strong promoter and achloramphenicol resistance marker. The fusion was made by SOE PCR(Horton, Hunt, Ho, Pullen, and Pease, 1989, Engineering hybrid geneswithout the use of restriction enzymes, gene splicing by overlapextension, Gene 77: 61-68). The SOE PCR method is also described in WO2003/095658. Each gene was expressed under the control of a triplepromoter system (as described in WO 99/43835), consisting of thepromoters from Bacillus licheniformis alpha-amylase gene (amyL),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillusthuringiensis cryIIIA promoter including stabilizing sequence. The genecoding for Chloramphenicol acetyl-transferase was used as marker.(Described in, e.g., Diderichsen, Poulsen, and Joergensen, 1993, Auseful cloning vector for Bacillus subtilis. Plasmid 30: 312). The finalgene construct was integrated on the Bacillus chromosome by homologousrecombination into the pectate lyase locus.

Chromosomal DNA of the four different Dyella japonica strains wasisolated by QIAmp Tissue Kit (Qiagen, Hilden, Germany). For each geneconstruct three fragments were PCR amplified: the gene fragment fromgenomic DNA from the 4 Dyella japonica species (all primers used arelisted in the Table 1 below), the upstream flanking fragment wasamplified with the primers #01-iMB1362 and iMB1362Uni2 and thedownstream flanking fragment was amplified with the primers #20-iMB1362and oth435 from genomic DNA of the strain iMB1362 (described in WO2003/095658).

The gene fragment was amplified using a proofreading polymerase(Phusion™ High-Fidelity DNA Polymerase, (New England Biolabs, Inc.)).The two flanking DNA fragments were amplified with “Expand High FidelityPCR System” (Roche-Applied-Science). The PCR reactions were madeaccording to standard procedures (following the manufacturer'srecommendations). The PCR conditions were as follows: 94° C. for 2minutes followed by 10 cycles of (94° C. for 15 seconds, 50° C. for 45seconds, 68° C. for 4 minutes) followed by 20 cycles of (94° C. for 15seconds, 50° C. for 45 seconds, 68° C. for 4 minutes (+20 seconds.extension per cycle)) and ending with one cycle at 68° C. for 10minutes. The 3 resulting fragments were mixed in equal molar ratios anda new PCR reaction were run under the following conditions: initial 2minutes at 94° C., followed by 10 cycles of (94° C. for 15 seconds, 50°C. for 45 seconds, 68° C. for 5 minutes), 10 cycles of (94° C. for 15seconds, 50° C. for 45 seconds, 68° C. for 8 minutes), 15 cycles of (94°C. for 15 seconds, 50° C. for 45 seconds, 68° C. for 8 minutes inaddition 20 seconds extra per cycle). After the 1^(st) cycle the two endprimers #01-iMB1362: and #20-iMB1362 were added (20 pMol of each). TwomicroL of the PCR product was transformed into Bacillus subtilis andtransformants was selected on LB-plates containing chloramphenicol (6microg/mL medium). A clone containing the construct without mutationsleading to amino acid changes was selected for fermentation in liquidmedia.

TABLE 1  Primers used Amplification of SPECIFIC PRIMER FORWARDSPECIFIC PRIMER REVERSE Dyella japonica 5′-CTTGCTGCCTCATTCTGCAG5′-GGGCCAAGGCCGGTTTTT (NN060814) gen 1 CCGCGGCCATCAACAGCATGACCTATGTTTTACTTCGAAATCAAC TTG-3′ (SEQ ID NO: 9) AACAACAGCG-3′(SEQ ID NO: 10) Dyella japonica 5′-CTTGCTGCCTCATTCTGCAG5′-GGGCCAAGGCCGGTTTTT (NN060812) gen 2 CCGCGACACCGGCCCAGGCGGCCTATGTTTTACTTGGAAATCAGC ATCAAC-3′ (SEQ ID NO: 11) AGCAGCAACGACTGGC-3′(SEQ ID NO: 12) Dyella japonica 5′-CTTGCTGCCTCATTCTGCAG5′-GGGCCAAGGCCGGTTTTT (NN060813) gen 3 CCGCGGCCATCAACAGCATGAGTTATGTTTTACTTGGAGATCAGC CTTG-3′ (SEQ ID NO. 13) AGCAACAGC-3′(SEQ ID NO: 14) Dyella japonica 5′-CTTGCTGCCTCATTCTGCAG5′-GGGCCAAGGCCGGTTTTT (NN060811) gen 4 CCGCGGCCATCAACAGCATGGGCTATGTTTTACTTCGAGATCAGC CT-3′ (SEQ ID NO: 15) AACAACAAAGACTG-3′(SEQ ID NO: 16) upstream flanking #01-iMB1362: iMB1362Uni2: fragment5′-ACAATATGCGGGACG-3′ 5′-CGCGGCTGCAGAATGAG (SEQ ID NO: 17)GCA-3′(SEQ ID NO: 18) downstream flanking oth435: #20-iMB1362: fragment5′-TAAAACATAAAAAACCGGCC 5′-GACATCAGCCCTGCT-3′ TTGGC-3′ (SEQ ID NO: 19)(SEQ ID NO: 20)Fermentation

The clones was streaked on an LB-agar plates with 6 micro g/mLchloramphenicol from −80° C. stock, and grown overnight at 37° C. Thecolonies were transferred to 100 mL Cal-18 media in a 500 mL shakingflask. The culture was shaken at 26° C. at 170 rpm for 2 or 3 days. Thecells were spun down and the enzyme purified from the supernatant bymethods described in Example 2.

Example 2

Purification of Dyella Isoamylase by Starch Affinity Chromatography

The crude ferment was filtered through a double filter paper sandwichlayer consisting of the following grades of Whatman filter paper, D, A,B, C and F with F at the bottom. This crude filtration was then followedby vacuum filtration with a vacuum cup filter with a 0.2 micro m poresize cut off.

The volume of filtrate was recorded and CaCl₂ was added under slowstirring to give a final concentration of 1 mM. To 900 mL of filtrateadd 100 mL of 1 M Sodium phosphate buffer pH 6.5 to give a finalconcentration of 0.1 M. It was checked that the pH was 6.5 and adjustedif necessary. The buffer addition may cause some precipitation, in thisevent the vacuum filtration step was repeated using a vacuum cup filterunit with a 0.2 micro m cut off.

Meanwhile a column was prepared (12×2.6 cm) with Amylose-agaroseaffinity column material (purchased from New England Biolabs)equilibrated with 0.1 M phosphate buffer pH 6.5. Load 200 mL of bufferedculture filtrate mixture on to the column (5 mLs/min) and collect theflow through. The column was washed with 0.1 M phosphate buffer untilthe UV absorbance was stable (the wash was collected). The isoamylasewas eluted from the column by washing the column with 0.1 M phosphatebuffer+20% Maltose. The peak was collected when the absorbance startedto climb over the base line level until the base line became stableagain (ca. 50-100 mL total volume). The column was reconditioned with0.1 M phosphate until a stable baseline was achieved and the load, wash,elute cycle was repeated until all the isoamylase supernatant was usedup. After the first run a 4-20% Tris-glycine SDS page gel was run tocheck for the presence of the isoamylase in the load, column flowthrough, column wash and in the eluted fractions.

20 microL of sample was mixed with 20 microL of sample buffer and heatedto 95° C. for 10 minutes. 20 microL was loaded to each lane and the gelwas run at 35 mA for one hour. Protein bands were realized using simplyBlue safe stain from Invitrogen. The Dyella isoamylase appeared as asingle band at 80 kD.

Once purity had been achieved the isoamylase fractions were pooled andwere then concentrated using Amicon Ultra ultracentrifuge units fittedwith a 30 kD cut off membrane (spun at 3,000 rpm×g for 30 minutes, thefiltrate was discarded and the load/concentrate cycle was continueduntil all the pooled fraction was used). Once concentrated, theconcentrate was washed several times with 0.1 M phosphate buffer pH 6.5to remove the 20% maltose. The concentrated isoamylase was removed witha pipette and saved. The inner membrane was washed several times with0.1 M phosphate buffer and each wash was pooled with the concentrate(this is important as unspecific binding will result in significantsample loss). The final volume was recorded.

The absorbance was measured at 280 nm using the phosphate buffer to zerothe spectrophotometer. If the absorbance was more than 2.0 the samplewas diluted in 0.1 M phosphate buffer so that the absorbance readingfailed within 0.5-1.5.

The protein concentration was calculated thus: A280 value/molarabsorbance (calculated from the amino acid sequence)×dilution factor.The value obtained was in mg/ml.

The sample was formulated by adding glycerol to a final concentration of50%.

Example 3

A spray-dried DE 11 maltodextrin, produced from common corn starchliquefied with Alpha-Amylase LS, was dissolved in deionized water andthe pH and solids level adjusted to approximately 4.3 and 30%respectively. Aliquots of the substrate containing 15 g dry solids weretransferred to 50 mL blue-cap flasks fitted with magnetic stirrers whichwere then placed in a water bath at 58° C. Different amounts ofGlucoamylase AN and Dyella japonica isoamylase (NN060812) were thenadded according to the scheme below and the pH's adjusted to 4.3.

Samples were taken at set time intervals and the reaction stopped byheating the sample to boiling. After dilution and sterile filtration thesamples were analyzed for D-glucose by HPLC.

Dyella Glucoamylase isoamylase Reaction time AGU/g DS IAU/g DS (hours) %D-glucose 0.18 0 24 91.45 0.18 0 48 95.88 0.18 0 72 96.35 0.12 100 2488.45 0.12 100 48 96.51 0.12 100 72 96.91

These data show that it is possible to obtain a higher D-glucose yieldwhen combining Dyella isoamylase with Aspergillus niger glucoamylase. Atthe same time, the glucoamylase dosage can be significantly reduced.

Example 4

A DE 11 maltodextrin substrate was prepared as described in Example 2.Different amounts of Glucoamylase AN, Glucoamylase T and Dyella japonicaisoamylase (NN060812) were added according to the scheme below. Thesaccharification reaction was carried out at 58° C., pH 4.3.

Samples were taken at set time intervals and the reaction stopped byheating the sample to boiling. After dilution and sterile filtration thesamples were analyzed for D-glucose by HPLC.

Dyella Reaction Glucoamylase AN Glucoamylase T isoamylase time % AGU/gDS AGU/g DS IAU/g DS (hours) D-glucose 0.18 0 0 24 91.45 0.18 0 0 4895.88 0.18 0 0 72 96.35 0 0.24 0 24 82.49 0 0.24 0 48 89.98 0 0.24 0 7292.72 0 0.24 100 24 89.27 0 0.24 100 48 94.67 0 0.24 100 72 95.59 0 0.24200 24 92.20 0 0.24 200 48 96.16 0 0.24 200 72 96.46

These data show that it is possible to match the performance ofGlucoamylase AN (Aspergillus niger glucoamylase) by using a dosage of0.24 AGU/g DS Glucoamylase T (Talaromyces emersonii glucoamylase) and100-200 IAU/g DS Dyella japonica isoamylase.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. An isolated polypeptide having isoamylaseactivity, which has at least 95% amino acid sequence identity to theamino acid sequence of amino acids 1-750 of SEQ ID NO:
 2. 2. Theisolated polypeptide of claim 1, having at least 96% amino acid sequenceidentity to the amino acid sequence of amino acids 1-750 of SEQ ID NO:2.
 3. The isolated polypeptide of claim 1, having at least 97% aminoacid sequence identity to the amino acid sequence of amino acids 1-750of SEQ ID NO:
 2. 4. The isolated polypeptide of claim 1, having at least98% amino acid sequence identity to the amino acid sequence of aminoacids 1-750 of SEQ ID NO:
 2. 5. The isolated polypeptide of claim 1,having at least 99% amino acid sequence identity to the amino acidsequence of amino acids 1-750 of SEQ ID NO:
 2. 6. The isolatedpolypeptide of claim 1, comprising or consisting of the amino acidsequence of SEQ ID NO:
 2. 7. The isolated polypeptide of claim 1,comprising or consisting of the amino acid sequence of amino acids 1-750of SEQ ID NO:
 2. 8. The isolated polypeptide of claim 1, which is afragment of the amino acid sequence of SEQ ID NO: 2 having isoamylaseactivity.
 9. A composition comprising the isolated polypeptide ofclaim
 1. 10. The composition of claim 9, further comprising aglucoamylase.
 11. The composition of claim 10, wherein the glucoamylaseis a Talaromyces emersonii glucoamylase.
 12. The composition of claim 9,further comprising one or more enzymes selected from the groupconsisting of alpha-amylases, beta-amylases, maltogenic amylases,alpha-glucosidases, pullulanases, hexosyltransferases, proteases, andbranching enzymes.
 13. A method for producing a glucose syrup,comprising (a) liquefying starch with an alpha-amylase; and (b) treatingthe liquefied starch with a glucoamylase and the polypeptide of claim 1to produce the glucose syrup.
 14. A method for producing maltose,comprising (a) liquefying starch with an alpha-amylase; (b) treating theliquefied starch with a beta-amylase and the polypeptide of claim 1 toproduce a maltose syrup; and (c) obtaining maltose from the maltosesyrup.
 15. A method for producing maltitol, comprising (a) liquefyingstarch with an alpha-amylase; (b) treating the liquefied starch with abeta-amylase and the polypeptide of claim 1 to produce a maltose syrup;(c) obtaining maltose from the maltose syrup; and (d) converting themaltose to maltitol.